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First published online 20 November 2002
doi: 10.1242/jcs.00237

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Increased Sp1 phosphorylation as a mechanism of hepatocyte growth factor (HGF/SF)-induced vascular endothelial growth factor (VEGF/VPF) transcription

¹ Department of Dermatology, Klinikum der J. W. Goethe-Universität, Frankfurt am Main, Germany
² Department of Molecular Biology, Max-Planck-Institut für Physiologische und Klinische Forschung, Bad Nauheim, Germany

View larger version (18K): [in a new window]   Fig. 1. HGF/SF does not regulate nuclear expression of Sp1 and Sp3 transcription factor in HaCaT cells. Representative western blot analysis of nuclear Sp1 and Sp3 expression by quiescent HaCaT cells, either left untreated (media change) or stimulated by HGF/SF for the indicated time periods. Total cellular protein (10 µg/lane) was separated by 10% SDS-PAGE, Sp1 and Sp3 protein were detected by enhanced chemiluminescence using specific antibodies to the respective nuclear factors (Santa Cruz; molecular weights are indicated to the right). Comparable results were obtained from three independent experiments.

View larger version (44K): [in a new window]   Fig. 2. HGF/SF neither changes relative DNA-binding activity of Sp1 and Sp3 factor nor induces additional Sp1-indendent binding to the -88/-65 bp VEGF/VPF promoter sequence. A representative EMSA using nuclear extracts of untreated (lanes 1, 3, 5, 7 and 9) or HGF/SF-stimulated HaCaT cells (10 minutes; lanes 2, 4, 6, 8 and 10). Unlabeled Sp1 consensus oligonucleotides (Promega) were used at a final concentration of 0.35 µM (lanes 3 and 4). Supershift analysis was performed by addition of specific Sp1 or/and Sp3 antibodies (Santa Cruz) at a final concentration of 100 ng/µl (lanes 5 to 10). The DNA sequence of the utilized probe is shown at the top (Sp1 sites underlined), the formation of Sp1-dependent binding complexes is indicated by arrows to the left; supershifted complexes are marked by a bold arrow to the right.

View larger version (24K): [in a new window]   Fig. 3. Sp1 and Sp3 transcription factor confer Sp1-site dependent VEGF/VPF promoter transactivation in a comparable fashion in Drosophila SL2 cells. Analysis of CAT expression derived from transiently transfected -88/+54 bp wild-type (wt) or -88/+54 Sp1 site-mutated (Sp1 mut) VEGF/VPF promoter-based construct (carrying critical 2 nucleotide mutations within the Sp1 sites; 5 µg each) along with expression vectors encoding Sp1 (pPacUSp1, 0.3 µg), Sp3 (pPacUSp3, 0.3 µg) and/or empty vector (pPacUbx, to compensate for differences in co-transfected DNA). The fold increase in CAT activity was calculated on the basis of data obtained from cells co-transfected with empty vector alone. Schematic representations of the wild-type and the mutated sequence are shown at the top (Sp1 sites underlined), the two nucleotide mutations are indicated by enlarged letter size. The data displayed represent the means±s.d. of five independent duplicate experiments. (Student's t-test; n.s., not significant; ** P<0.01, compared to cells transfected with the wt vector).

View larger version (17K): [in a new window]   Fig. 4. HGF/SF increases cellular amounts of serine-phosphorylated transcription factor Sp1. Western blot analyses of HaCaT cells that were left untreated (media change) or were stimulated by HGF/SF (10 minutes at 100 ng/ml). Cellular extracts were immunoprecipitated (IP) by a specific anti-Sp1 antibody (Sigma) prior to immunoblotting (IB). (A) Immunoblotting with Sp1 antibody (upper panel) and phospho-serine monoclonal antibody (clone 4A3; lower panel). (B) Immunoblotting with Sp1 antibody (upper panel) and phospho-threonine antibody (lower panel). The immunoblots displayed are representative of three that were performed revealing comparable results.

View larger version (14K): [in a new window]   Fig. 5. Pharmacological inhibition of conventional and novel PKC isoforms blocks HGF/SF-induced PKC phosphorylation but fails to inhibit HGF/SF-mediated VEGF/VPF gene expression. (A) Detection of phosphorylated PKC- (p-PKC-, upper panel) or the respective actin protein expression (from Santa Cruz) by western blot analysis. HaCaT cells were left untreated or were exposed to HGF/SF (for 10 minutes at 100 ng/ml) after preincubation with broad-range PKC inhibitor calphostin C (Cal. C at 1 µM for 60 minutes), isotype-selective (conventional and novel) PKC inhibitor bisindolylmaleimide I (Bis. I at 1 µM for 60 minutes) or solvent only (DMSO, 0.1%) as indicated. Experiments were repeated twice with similar results. (B) Analysis of CAT expression derived from a transiently transfected -88/+54 bp VEGF/VPF-promoter-based construct. HaCaT cells were cultured in the absence or presence of bisindolylmaleimide I (Bis. I at 1 µM, starting 1 hour prior to HGF/SF treatment) without growth factor stimulation or with HGF/SF treatment (for 16 hours at 100 ng/ml) as indicated. Data displayed represent the means±s.d. of three independent triplicate assays. (C) VEGF/VPF protein of supernatants derived from confluent HaCaT cells. Cells were cultured in the absence or presence of calphostin C (at 1 µM, starting 1 hour prior to HGF/SF treatment) without growth factor stimulation or with HGF/SF treatment (for 24 hours at 100 ng/ml) as indicated. Data from three independent triplicate experiments are expressed as ng secreted VEGF/VPF protein per mg total cellular protein (mean±s.e.m.).

View larger version (14K): [in a new window]   Fig. 6. Targeting PKC- expression attenuates HGF/SF-induced VEGF/VPF promoter activity. (A) Western blot analyses of HaCaT cells that were left untreated (media change) or were stimulated by HGF/SF (10 minutes at 100 ng/ml). Cellular extracts were immunoprecipitated (IP) by a specific anti-PKC- antibody (Santa Cruz) prior to immunoblotting (IB). Immunoblotting with pan-PKC- antibody (Santa Cruz; upper panel) and with phospho-PKC- antibody (lower panel). (B) Analysis of firefly luciferase (Luc) expression derived from a transiently transfected -88/+54 bp VEGF/VPF-promoter-based reporter construct (1 µg) along with antisense oligonucleotides directed against the translation start site of PKC- (second bar) or of PKC- (third bar, each at 0.1 µM). Twenty-four hours after transfection, HaCaT cells were stimulated with HGF/SF (at 100 ng/ml) for 16 hours or were left untreated. This assay is representative of three independent sets of experiments revealing comparable results. Fold increase in HGF/SF-induced luciferase activity is calculated on the basis of data obtained from the respective controls, which were left unstimulated. Values represent the mean±s.d. of triplicate assays. Statistical analyses were performed on data from three experiments (Student's t-test, *P<0.05). (C) Analysis of firefly luciferase expression derived from a transiently transfected -88/+54 bp VEGF/VPF-promoter-based reporter construct (2.5 µg) along with wild-type PKC- (wt-PKC-) or its kinase-deficient mutant (mut-PKC-; 500 ng each) vector. Twenty-four hours after transfection, HaCaT cells were stimulated with HGF/SF (at 100 ng/ml) for 16 hours or were left untreated. Data displayed herein include the values of three independent duplicate experiments (mean±s.e.m.; Student's t-test, *P<0.05).

View larger version (15K): [in a new window]   Fig. 7. Chemical inhibition of the PI 3-kinase pathway blocks HGF/SF-induced Akt phosphorylation and inhibits HGF/SF-mediated VEGF/VPF gene transcription and protein expression. (A) Detection of phosphorylated Akt (at residue threonine 308, Thr308; upper panel; at serine residue 473, Ser473, lower panel) or of the respective total Akt protein expression (Akt; antibodies from Cell Signaling) by western blot analysis. HaCaT cells were left untreated or were exposed to HGF/SF (for 10 minutes at 100 ng/ml) after preincubation with the PI 3-kinase inhibitors wortmannin (Wort., at 100 nM, for 60 min) or LY 294002 (LY; at 10 µM, for 60 min) or with solvent only (DMSO, 0.1%) as indicated. Experiments were repeated three times with comparable results. (B) Analysis of CAT expression derived from a transiently transfected -88/+54 bp VEGF/VPF-promoter-based construct. HaCaT cells were cultured in the absence or presence of wortmannin (at 100 nM, starting 1 hour prior to HGF/SF treatment) without growth factor stimulation or with HGF/SF treatment (for 16 hour at 100 ng/ml) as indicated. Values represent the mean±s.d. of three triplicate assays. (C) VEGF/VPF protein of supernatants derived from confluent HaCaT cells. Cells were cultured in the absence or presence of LY 294002 (at 10 µM, for 60 minutes, starting 1 hour prior to HGF/SF treatment) without growth factor stimulation or with HGF/SF treatment (for 24 hours at 100 ng/ml) as indicated. Data from three triplicate experiments are expressed as ng secreted VEGF/VPF protein per mg total cellular protein (mean±s.e.m.). Statistical analyses were performed on data from three sets of experiments (Student's t test, **P<0.01, *P<0.05).

View larger version (12K): [in a new window]   Fig. 8. Inhibition of MEK1/2 blocks HGF/SF-induced PKC phosphorylation of ERK1/2 and inhibits HGF/SF-mediated VEGF/VPF gene transcription and protein expression. (A) Detection of phosphorylated ERK1/2 (p-ERK1/2, upper panel) or total ERK1/2 protein (ERK1/2, lower panel) by western blot analysis. HaCaT cells were left untreated or were exposed to HGF/SF (for 10 minutes at 100 ng/ml) after preincubation with the MEK1/2 inhibitor PD 98059 (at 50 µM for 60 minutes) or solvent only (DMSO, 0.1%) as indicated. Experiments were repeated three times with comparable results. (B) Analysis of CAT expression derived from a transiently transfected -88/+54 bp VEGF/VPF-promoter-based construct. HaCaT cells were cultured in the absence or presence of PD 98059 (at 50 µM, starting 1 hour prior to HGF/SF treatment) without growth factor stimulation or with HGF/SF treatment (for 16 hours at 100 ng/ml) as indicated. The data displayed represent the mean±s.d. of three triplicate assays. (C) VEGF/VPF protein of supernatants derived from confluent HaCaT cells. Cells were cultured in the absence or presence of PD 98059 (at 50 µM, starting 1 hour prior to HGF/SF treatment) without growth factor stimulation or with HGF/SF treatment (for 24 hours at 100 ng/ml) as indicated. Data from three triplicate experiments are expressed as ng secreted VEGF/VPF protein per mg total cellular protein (mean±s.e.m.). Statistical analyses were performed on data from three sets of experiments (Student's t-test, **P<0.01, *P<0.05).

View larger version (32K): [in a new window]   Fig. 9. Effects of MEK1 (PD 98059) or PI 3-kinase (LY 294002) inhibition on HGF/SF-induced phosphorylation of Akt and ERK1/2 in HaCaT keratinocytes. (A) Detection of phosphorylated Akt (at residue serine 473, Ser473, upper panel) or detection of phosphorylated ERK1/2 (p-ERK1/2, lower panel, antibodies from Cell Signaling) by western blot analysis. HaCaT cells were left untreated or were exposed to HGF/SF (for 10 minutes at 100 ng/ml) after preincubation with PI 3-kinase inhibitor LY 294002 (LY; at 10 µM for 60 minutes), with the MEK1 inhibitor PD 98059 (PD; at 50 µM for 60 minutes) or with solvent only (DMSO, 0.1%) as indicated. (B) Detection of phosphorylated Akt or ERK1/2 in HaCaT cells that were either transiently transfected with parent vector only (pCMV; lanes 1 and 2) or with dominant-negative PKC- mutant (pCMV PKC-MUT; 500 ng each). Thirty-eight hours after transfection, cells were left untreated or were exposed to HGF/SF (for 10 minutes at 100 ng/ml). Experiments were repeated twice with comparable results.

View larger version (31K): [in a new window]   Fig. 10. Inhibition of PI 3-kinase (LY 294002) and MEK1 (PD 98059) prevents HGF/SF-induced phosphorylation of PKC-. Western blot analyses of HaCaT cells that were left untreated (media change) or were exposed to HGF/SF (for 10 minutes at 100 ng/ml) after preincubation with (A) PI 3-kinase inhibitor LY 294002 (LY; at 10 µM for 60 minutes), with (B) MEK1 inhibitor PD 98059 (PD; at 50 µM for 60 minutes) or with solvent only (DMSO, 0.1%). Cellular extracts were immunoprecipitated (IP) by specific anti-PKC- antibody (Santa Cruz) prior to immunoblotting (IB). Immunoblotting with pan-PKC- antibody (upper panel) and with phospho-PKC- antibody (lower panel). Immunoblots displayed are representative of two that were performed revealing comparable results.

View larger version (35K): [in a new window]   Fig. 11. HGF/SF-induced serine-phosphorylation of Sp1 is attenuated by PI 3-kinase, MEK1 and broad range PKC inhibition. Western blot analyses of HaCaT cells that were left untreated (media change, lane 1) or were stimulated by HGF/SF (for 10 minutes at 100 ng/ml) after preincubation with MEK1 inhibitor PD 98059 (PD; at 50 µM for 60 minutes), with PI 3-kinase inhibitor LY 294002 (LY; at 10 µM, for 60 minutes), with broad range PKC inhibitor RO 31-8220 (RO; at 20 µM for 60 minutes) or with solvent only (DMSO, 0.1%, lane 2). Cellular extracts were immunoprecipitated (IP) by specific anti-Sp1 antibody (Sigma) prior to immunoblotting (IB). Immunoblotting with a Sp1 antibody (upper panel) and with a phospho-serine monoclonal antibody (Biomol, clone 4A3; lower panel). Experiments were repeated twice with comparable results.